RESEARCH ARTICLE W. Sun, J-Y. Han, Q-J. Li, K.

Jiao, 42
S. Afr. J. Chem., 2007, 60, 42–46,
<http://journals.sabinet.co.za/sajchem/>.

Spectrophotometric and Voltammetric Studies on the

Interaction of Heparin with Crystal Violet and its
Analytical Application
Wei Sun*, Jun-Ying Han, Qing-Jun Li and Kui Jiao
College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China.

Received 3 May 2005; revised 22 September 2006; accepted 14 May 2007.

ABSTRACT
The interactions of heparin with crystal violet were studied by absorption spectrophotometry and voltammetry in pH 3.0
Britton-Robinson (B-R) buffer solution. Heparin, which is negatively charged, can easily bind to the positively charged crystal
violet to form a supramolecular ion-association complex. Owing to the formation of the new complex, the maximum absorption
wavelength of crystal violet at 592 nm decreased and two new absorption peaks appeared at 510 nm and 363 nm after the addition
of heparin. The oxidation peak current of crystal violet on glassy carbon electrode (GCE) at + 0.84 V (vs. SCE) also changed corre-
spondingly without change of the peak potential, which also indicated that the binding reaction had taken place. Under the
selected conditions a new spectrophotometric analytical method was established for heparin with the linear range between
0.10–4.0 mg L–1 with a linear regression equation as ∆A = –0.002 + 0.227C (mg L–1), (n = 8, γ = 0.997). The relative standard
deviation for eleven parallel determinations of 0.40 mg L–1 heparin was 1.69% and the detection limit (3σ) was 0.09 mg L–1. This new
method was further applied to the determination of heparin sodium injection samples with satisfactory results.
KEYWORDS
Heparin, crystal violet, spectrophotometry, voltammetry, ion-association complex.

1. Introduction cationic dyes to form a heparin-dye supramolecular ion-associa-

Heparin is an important biochemical medicine. It has many
biological functions such as anticoagulant, antithrombotic,
antilipemic and antiatherosclerosis activities.1 Research has
shown that it has potential therapeutic functions. For example,
heparin can interact with many biological proteins such as
proinflammatory chemokines, growth factors, extracellular
matrix proteins and platelets.2,3 According to its structure,
heparin is a polysaccharide of the glycosaminoglycan (GAG)
family, which has linear and strongly negatively charged sugar
chains of repeating disaccharide units of duronic/glucuronic
acid and glucosamine residues. The molecular mass of commer-
cial heparin products ranges from 3000 to 30 000 Da with an
average of 12 000 Da.
Many different methods have been proposed for the assay of
GAGs4–7 and heparin such as a biological method,8 UV-Vis
spectrophotometry,9 light-scattering technique,10 HPLC and
electrophoresis.11,12 Among them the most widely used method is
UV-Vis spectrophotometry based on the metachromatic
interactions with cationic dyes such as toluidine blue, methylene
blue and azure A. Jiao et al. have applied methylene blue and
azure A as spectrophotometric probes for the detection of
heparin and investigated the binding mechanism.13 Recently
Liu et al. reported the interaction of heparin with some basic
diphenylnaphthylmethane dyes such as victoria blue 4R, victoria
blue B, night blue and others by resonance Rayleigh scattering
technique or fading spectrophotometry.10,14,15 Owing to the
presence of three O-sulphate groups, two N-sulphate groups
and two carboxyl groups per tetrasaccharide unit of heparin, it
has a high anionic charge density. Therefore it can interact with
tion complex.
In this paper crystal violet was selected as the bioprobe to
investigate the binding reaction with heparin and further used
for the detection of micro-amounts of heparin. It is a kind of
basic triphenylmethane dye commonly used in the analytical
laboratory and can easily be obtained at low costs. Crystal violet
which is a cationic dye (molecular structure shown in Fig. 1) has
been used for the detection of metal ions. Zhang et al. have
applied crystal violet for the detection of nucleic acids by reso-
nance light-scattering technique based on its interaction with
DNA.16 In pH 3.0 Britton-Robinson (B-R) buffer solutions, the
O-sulphate and N-sulphate groups in the structures of heparin
completely dissociate. The heparin molecule is negatively
charged and can easily interact with cationic dyes. Based on
this principle, the interaction of heparin with crystal violet was
investigated by spectrophotometry and electrochemical meth-
ods and further applied to the detection of heparin in heparin
sodium injection samples.

* To whom correspondence should be addressed. E-mail: sunwei@qust.edu.cn Figure 1 Molecular structure of crystal violet.
RESEARCH ARTICLE W. Sun, J-Y. Han, Q-J. Li, K. Jiao, 43
S. Afr. J. Chem., 2007, 60, 42–46,
<http://journals.sabinet.co.za/sajchem/>.

Figure 2 UV-Vis absorption spectra of different concentrations of crystal violet (a) and the interaction of crystal violet with different amounts of
heparin (b) in pH 3.0 B-R buffer solution. (a) 1 → 5: 0.5, 1.0, 1.5, 2.5, 3.5 × 10–5 mol L–1 crystal violet; (b) 1: 2.5 × 10–5 mol L–1 crystal violet; 2 → 5: 1 + 1.0, 2.0,
3.0, 10.0 mg L–1 heparin.

2. Experimental ate amount of heparin solution or heparin injection sample

2.1. Apparatus
A Cary 50 probe spectrophotometer (Varian, Australia) and a
model 752 UV-Vis spectrophotometer (Shanghai No. 3 Analyti-
cal Instrument Factory, China) were used for recording absorp-
tion spectra or measuring the absorbance at a fixed wavelength,
using a 1-cm pathlength cell. Cyclic voltammetric experiments
were carried out using a model CHI 832 electrochemical
analyser (Shanghai CH Instrument, China) with a glassy carbon
electrode (GCE) (Φ = 3.0 mm) as working electrode, a saturated
calomel reference electrode (SCE) and a platinum wire auxiliary
electrode. pH values were measured with a pHS-25 acidity meter
(Shanghai Leici Instrument Factory, China). All experiments were
carried out at 25 ± 2 °C.
2.2. Reagents
Heparin (sodium salt, 140 IU mg–1, Shanghai Chemical Reagent
Plant) was used as received without further purification. The
1.0 mg mL–1 stock solution of heparin (140 IU mL–1) was prepared
by directly dissolving 0.1000 g of heparin sodium reagent in
doubly distilled water from all-quartz still, diluted to 100 mL and
stored at 4 °C. The working solutions were obtained by diluting
the stock solution with water. 1.0 × 10–3 mol L–1 crystal violet
(Zhongguo Yuanhang Chemical Reagent Factory) was prepared
by dissolving 0.04080 g crystal violet in water and diluting to
100 mL. 0.2 mol L–1 Britton-Robinson (B-R) buffer solution was
used to control the acidity of the reaction solution, which was
prepared by mixing 12.35 g boric acid, 13.55 mL 85 % phosphoric
acid and 11.80 mL acetic acid diluted to 1000 mL and adjusted to
pH 3.0 by 0.2 mol L–1 aqueous solution of sodium hydroxide.
All other reagents were of analytical reagent grade and were
used without further purification. Doubly distilled water was
used throughout.
2.3. Procedure
To a dry 10 mL colorimetric tube, solutions were added in
the following order: 1.5 mL of 0.2 mol L–1 B-R (pH 3.0) buffer
solution, 2.5 mL 1.0 × 10–4 mol L–1 crystal violet and an appropri-
solution. The mixtures were diluted to 10 mL with water, mixed
homogeneously and allowed to stand for 10 min at 25 °C.
For spectrophotometric detection, the absorbances of the
solution were recorded at 592 nm against water. The absorbance
(A0) of the blank sample without heparin was obtained under
the same conditions, and thus the difference of absorbance
(∆A = A0–A) was obtained for measurement. For electrochemical
measurements the cyclic voltammetric curves of crystal violet
and its mixture with heparin were recorded on GCE and
compared with each other in the potential sweep range of
0.5–1.1 V (vs. SCE). The changes of peak current of crystal violet
were compared.
The reaction conditions were optimized for the spectrophoto-
metric method. The effect of pH on the absorbance was investi-
gated at pH 1.4–4.5 by using different B-R buffers to control the
acidity of the reaction solution. The effect of crystal violet
concentration on the absorbance was studied with 5.0 mg L–1
heparin solutions using different crystal violet concentrations in
the range of 0.5 × 10–5–5.0 × 10–5 mol L–1. The reaction time was
examined by measurement of the absorbance of crystal violet-
heparin reaction solution at 592 nm for about 3 h immediately
after mixing. The influences of various substances were studied
by premixing interfering substances with 2.0 mg L–1 heparin
solution using the general spectrophotometric procedure.
3. Results and Discussion
3.1. UV-Vis Absorption Spectra of the Crystal Violet–Heparin
Interaction System
The absorption spectra of different concentrations of crystal
violet in pH 3.0 B-R buffer solution were scanned in the range
of 300–700 nm as shown in Fig. 2(a). It can be seen that the
maximum absorbance peak appeared at 592 nm and with the
increase of the concentration of crystal violet, the absorbance at
592 nm increased correspondingly.
The UV-Vis absorption spectra of crystal violet and its mixture
with heparin in pH 3.0 B-R buffer solutions are shown in
Fig. 2(b), which were obtained by keeping the crystal violet
RESEARCH ARTICLE W. Sun, J-Y. Han, Q-J. Li, K. Jiao, 44
S. Afr. J. Chem., 2007, 60, 42–46,
<http://journals.sabinet.co.za/sajchem/>.

Figure 3 Cyclic voltammograms of different concentration of crystal violet (a) and the interaction of crystal violet with different amounts of heparin
(b) in pH 3.0 B-R buffer solution. (a) 1 → 5: 0.5, 1.0, 1.5, 2.5, 3.5 × 10–5 mol L–1 crystal violet; (b) 1: 2.5 × 10–5 mol L–1 crystal violet; 2 → 4: 1 + 1.0, 3.0,
10.0 mg L–1 heparin. Scan rate: 50 mV s–1.

concentration and pH value constant and changing the concen- pH 3.0 B-R buffer solution were recorded and are shown in
tration of heparin. In the wavelength range from 300 to 700 nm, Fig. 3(b). Crystal violet had an irreversible oxidative peak at
heparin showed no absorbance and crystal violet has a maximum +0.84 V (vs. SCE) and after the addition of heparin, the oxidative
absorption at 592 nm (curve 1). On the addition of heparin, the peak current of crystal violet increased without change of the
absorption peak of crystal violet at 592 nm decreased while new peak potential. No new peak appeared on the cyclic voltammo-
absorption peaks appeared at 510 nm and 363 nm (curves 2–4), gram of crystal violet-heparin reaction solutions. We therefore
which were attributed to the formation of heparin-crystal violet conclude that under the selected conditions a supramolecular
complex. The absorption peak of the complex formed was ion-association complex was formed, which caused the changes
apparently different from that of crystal violet because the of the peak current. The more heparin was added, the more
wavelengths corresponding to both absorbance maxima were the peak currents changed. The changes of electrochemical
different. A well-defined isobestic point is formed at 524 nm. The responses of crystal violet in the presence and absence of hepa-
decrease of absorbance value at 592 nm was proportional to the rin also demonstrate the interaction of crystal violet with hepa-
heparin concentration and can be further applied to the detection rin to form a crystal violet-heparin complex.
of heparin samples.
3.3. Optimization of Spectrophotometry Parameters
3.2. Cyclic Voltammograms of the Crystal Violet–Heparin
Interaction System 3.3.1. Effects of pH and Buffers
The cyclic voltammetric curves of different concentrations of The difference of absorbance values (∆A) was greatly affected
crystal violet in pH 3.0 B-R buffer solution were also recorded by the pH of the buffer solution and the influence of pH was
and the results are shown in Fig. 3(a). It can be seen that crystal investigated in the pH range of 1.4 to 4.5. As shown in Fig. 4, ∆A
violet had an oxidative peak at +0.84 V (vs. SCE) and did not reached its maximum at pH 3.0. Therefore this pH was chosen
have any reductive peaks, which indicated that the electro- for the assay. At this pH, crystal violet was positively charged
chemical behaviour of crystal violet on GCE was irreversible in
pH 3.0 B-R buffer solution. The multiple sweep cyclic
voltammetric experiments showed that with the increase of
scanning time, the oxidative peak current of crystal violet
decreased correspondingly, which was due to the accumulation
of the oxidative product of crystal violet on the surface of GCE to
form an insulated membrane which interfered with the transfer
of electrons. The peak potential of crystal violet shifted negatively
with the increase of the pH value of buffer solution, which
shows that the electrode reaction involves protons. The peak
current of crystal violet increased with the increase of the scan-
ning rate and the plot of the oxidative peak current against the
square root of scanning rate was linear in the range from 200 mV
s–1 to 800 mV s–1 with a correlation coefficient of γ = 0.996, which
indicated that the electrode reaction was controlled by diffusion
processes.
Figure 4 Influence of pH on the crystal violet-heparin interaction 1.5 mL
The cyclic voltammograms of 2.5 × 10–5 mol L–1 crystal violet different pH of B-R buffer +2.0 × 10–5 mol L–1 crystal violet + 20.0 mg L–1
solution and its interaction with different amounts of heparin in heparin.
RESEARCH ARTICLE W. Sun, J-Y. Han, Q-J. Li, K. Jiao, 45
S. Afr. J. Chem., 2007, 60, 42–46,
<http://journals.sabinet.co.za/sajchem/>.

and heparin negatively charged, so they can strongly interact

with each other to form a supramolecular complex. The nature
of the buffer also affected the interaction and different kinds of
buffers such as B-R, HOAc-NaOAc, NH3-NH4Cl and others were
tested. In B-R buffer solution the response was maximal. For this
reason B-R buffer was used in this paper. The volume range of
0.5–4.0 mL of a 0.2 mol L–1 B-R buffer was investigated and the
addition of 1.5 mL was selected for the following procedures.

3.3.2. Effect of Crystal Violet Concentration

The concentration of crystal violet affected the difference of
absorbance values. ∆A increased with the increase of the concen-
tration of crystal violet in the range of 0.5 × 10–5–2.5 × 10–5 mol L–1
and remained constant with further increase of concentration,
which indicated that the binding reaction had reached its
equilibrium. Therefore a concentration of 2.5 × 10–5 mol L–1 of
crystal violet was recommended for use in this paper.
3.3.3. Reaction Time and Temperature
The stability of the reaction solutions was investigated. After
mixing heparin with crystal violet, the binding reaction
occurred rapidly. The absorbance differences reached a maxi-
mum within 20 min and remained unchanged for at least 2 h.
Therefore, this system gave enough time for routine measure-
ments.
The effect of the reaction temperature on the interaction was
tested at 15 °C, 25 °C, 30 °C and 37 °C, respectively, and found to
be insignificant. A reaction temperature of 25 °C was therefore
used throughout.
3.4. Effect of Coexisting Substances
Some substances such as metal ions, amino acids, glucose etc.
may exist in biological samples. The possible interferences on
the determination were studied by mixing them with 2.0 mg L–1
heparin solution. The results are listed in Table 1 and it can be
seen that few substances interfere with this assay and good
selectivity can be obtained. This method can therefore be
applied for the direct determination of heparin in heparin
sodium injection samples.
The influences of some surfactants such as sodium dodecyl
sulphate (SDS), Tween-20, OP-10, β-cyclodextrin (β-CD),
cetyltrimethylammonium bromide (CTAB) and others on the
binding reaction were also investigated. The addition of
non-ionic surfactants such as Tween-20, OP-10 and β-CD seldom
affected the binding reaction, but the addition of SDS and CTAB
Figure 5 Influence of ionic strength on the difference of absorbance
value 2.0 × 10–5 mol L–1 crystal violet and 2.0 mg L–1 heparin in pH 3.0 B-R
buffer solution.
can greatly influence the value of ∆A, which may be explained
by the dissociation of the ionic surfactants in solution and the
reaction with heparin to form a surfactant-heparin complex.
The effect of NaCl concentration in the range of 0.1–0.6 mol L–1
on this assay was also examined. The results demonstrate
the significant influence of NaCl on the heparin–crystal violet
interaction (shown in Fig. 5). The ∆A decreased with increasing
salt concentration, which indicated that the interaction of crystal
violet with heparin was mainly caused by electrostatic attrac-
tion. The increase of Na+ concentration caused an increasing
electrostatic shielding effect and thereby decreased the formation
of heparin-crystal violet complex resulting in the decrease of the
absorbance signal.
3.5. Calibration Curve for Heparin
Under optimal conditions, a calibration curve was obtained
between ∆A at 592 nm and the concentration of heparin with
2.5 × 10–5 mol L–1 crystal violet. Good linearity was obtained in
the concentration range of 0.10–4.0 mg L–1 with the linear regres-
sion equation ∆A = –0.002 + 0.227C (mg L–1), (n = 8, γ = 0.997).
The relative standard deviation for eleven parallel determina-
tion of 0.40 mg L–1 heparin was 1.69% and the detection limit (3σ)
was 0.09 mg L–1. The sensitivity of the crystal violet spectropho-
tometric method for heparin determination is sufficient for
routine detection.

Table 1 Influence of coexisting substances on the determination of 2.0 mg L–1 heparin.

Coexising substances Concentration Relative error Coexisting substances Concentration Relative error
/mol L–1 /% /mg L–1 /%

Ba2+ 1.0 × 10–5 3.45 L-Leucine 10.0 3.05

Fe3+ 1.0 × 10–5 –1.15 L-Lysine 10.0 3.95
Cd2+ 1.0 × 10–5 –1.15 L-Valine 10.0 –0.72
Ni2+ 1.0 × 10–5 –0.99 L-Glutamic acid 10.0 1.80
Hg2+ 1.0 × 10–5 1.64 L-Arginine 10.0 1.97
Zn2+ 1.0 × 10–5 1.48 L-Cysteine 10.0 2.49
Co2+ 1.0 × 10–5 0.66 L-Tryptophan 10.0 5.18
Ca2+ 1.0 × 10–5 –1.82 L-Serine 10.0 –1.71
OP-10 1.0 × 10–5 –4.52 Citric acid 5.0 4.31
Tween 20 1.0 × 10–5 5.17 Glucose 5.0 4.94
β-CD 1.0 × 10–5 4.93 Alcohol 5.0 –3.98
SDS 1.0 × 10–5 32.16 Urea 5.0 4.20
CTAB 1.0 × 10–5 20.74 HSA 1.0 3.68

β-CD, β-cyclodextrin; SDS, sodium dodecyl sulphate; CTAB, cetyltrimethylammonium bromide; HSA, human serum albumin.
RESEARCH ARTICLE W. Sun, J-Y. Han, Q-J. Li, K. Jiao,
S. Afr. J. Chem., 2007, 60, 42–46,
<http://journals.sabinet.co.za/sajchem/>.
Table 2 Determination of heparin in heparin sodium injection sample and recovery.
Sample no. Specified Detected
/mg L–1 /mg L–1
020911-1 0.893 0.888
20031003 0.893 0.875
Figure 6 Molecular structure of heparin.
3.6. Sample Determination and Recovery Test
The heparin sodium injection samples were purchased from
Anhui Xinli Pharmaceutical Company of China (sample no.
020911-1) and Tianjing Biochemical Pharmaceutical Factory of
China (sample no. 20031003) with the specified amount of
heparin as 6250 IU mL–1. The procedure for sample assay was as
follows: a 1.0 mL portion of heparin sodium injection was
pipetted into a 100 mL calibrated flask and was diluted to the
mark with water. A 1.0 mL amount of this solution was further
pipetted into a 100-mL volumetric flask and then diluted to the
mark with water. A 2.0 mL portion of this solution was used in
the general procedure with spectrophotometric detection. The
results of determinations and recovery tests are listed in Table 2.
It can be seen that this new spectrophotometric method was
practical and reliable.
3.7. Discussion of the Interaction Mechanism
Heparin is a polysaccharide of the glycosaminoglycan (GAG)
family consisting of repeating sulphated and/or carboxylated
disaccharide units in its molecular structure, which has three
O-sulphate, two N-sulphate and two carboxyl groups per tetra-
saccharide unit. The sulphate groups completely dissociate even
below pH 3.0. The carboxyl group is weakly acidic and the pKa
of D-glucuronic acid in heparin is 3.6. The carboxyl groups
gradually dissociate with an increase of pH and with complete
dissociation above pH 5.0.17 Owing to the presence of sulphate
and carboxyl groups and under the selected conditions, heparin
exists in a polyvalent anionic state with the anionic groups being
mainly –OSO3–, –NHSO3– and –COO– (as shown in Fig. 6)
and with the tetrasaccharide groups linked via oxo bridges. In
pH 3.0 B-R buffer solution, the O-sulphate and N-sulphate
46
RSD Added Found Recovery
/% /mg L–1 /mg L–1 /%
1.10 2.00 2.070 103.5
3.43 2.00 1.954 97.7
groups completely dissociate and the whole heparin molecule is
negatively charged, while the cationic dye crystal violet species
is positively charged. The two species bind via electrostatic
forces and hydrophobic interaction to form a supramolecular
complex, which results in the changes of the spectrophotometric
and voltammetric responses of the reaction system. The change
in absorbance of the heparin-crystal violet reaction system at
592 nm can be further used to establish a new spectrophotomet-
ric method for the detection of micro amounts of heparin and
applied to heparin injection samples with satisfactory results.
Acknowledgements
This project was financially supported by the National Natural
Science Foundation of China (20405008, 20635020), the Natural
Science Foundation of Qingdao City (04-2-JZ-114) and Doctoral
Foundation of QUST (0022125).
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